(EMSA) for Measuring Dissociation Constants of Protein‐RNA Complexes

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Label-Free Electrophoretic Mobility Shift Assay (EMSA) for Measuring Dissociation Constants of Protein-RNA Complexes Minguk Seo,1 Li Lei,1 and Martin Egli1,2 1Department of Biochemistry, School of Medicine, Vanderbilt University, Nashville, Tennessee 2Corresponding author: [email protected]

The electrophoretic mobility shift assay (EMSA) is a well-established method to detect formation of complexes between proteins and nucleic acids and to determine, among other parameters, equilibrium constants for the interaction. Mixtures of protein and nucleic acid solutions of various ratios are analyzed via polyacrylamide gel electrophoresis (PAGE) under native conditions. In general, protein–nucleic acid complexes will migrate more slowly than the free nucleic acid. From the distributions of the nucleic acid components in the observed bands in individual gel lanes, quantitative parameters such as the dissociation constant (Kd) of the interaction can be measured. This article describes a simple and rapid EMSA that relies either on precast commercial or handcast polyacrylamide gels and uses unlabeled protein and nucleic acid. Nucleic acids are instead detected with SYBR Gold stain and band intensities established with a standard gel imaging system. We used this protocol speciﬁcally to determine Kd values for complexes between the PAZ domain of Argonaute 2 (Ago2) enzyme and native and chemically modiﬁed RNA oligonucleotides. EMSA- based equilibrium constants are compared to those determined with isothermal titration calorimetry (ITC). Advantages and limitations of this simple EMSA are discussed by comparing it to other techniques used for determination of equilibrium constants of protein-RNA interactions, and a troubleshooting guide is provided. C 2018 by John Wiley & Sons, Inc. Keywords: Ago2 r electrophoresis r equilibrium constant r protein–nucleic acid interaction r RNA

How to cite this article: Seo, M., Lei, L., & Egli, M. (2019). Label-free electrophoretic mobility shift assay (EMSA) for measuring dissociation constants of protein-RNA complexes. Current Protocols in Nucleic Acid Chemistry, 76, e70. doi: 10.1002/cpnc.70

Nucleic acid–protein interactions are ubiquitous and absolutely essential in biological BASIC PROTOCOL information transfer, including replication, transcription, repair, and RNA metabolism (Cusack et al., 2017). Gel electrophoresis using polyacrylamide gels (PAGE; Andrus & Kuimelis, 2001a) and the electrophoretic mobility shift assay (EMSA) remain important and are widely used approaches for detecting nucleic acid–protein interactions and determining the stability of individual complexes (Alves & Cunha, 2012; Chen, 2011; Fried, 1989; Hellman & Fried, 2007). This protocol details the steps for establishing the equilibrium dissociation constant Kd of an RNA-protein complex by EMSA. Solutions of RNA and protein are mixed in different ratios, and the

Seo et al.

Current Protocols in Nucleic Acid Chemistry e70, Volume 76 1of12 Published in Wiley Online Library (wileyonlinelibrary.com). doi: 10.1002/cpnc.70 C 2018 John Wiley & Sons, Inc. binding reactions are then separated by non-denaturing PAGE (https://tools.thermofisher. com/content/sfs/brochures/1601945-Protein-Interactions-Handbook.pdf). In general, a protein-RNA complex will migrate more slowly through the gel matrix relative to the RNA alone, thus causing a shift on the gel (e.g., Yang et al., 1999). Individual bands can be visualized by end-labeling the RNA radioactively (32P) or by using fluorescent or chemiluminescent probes in combination with a gel imager. However, in our assay, we have used non-labeled RNA together with the SYBR Gold stain (Tuma et al., 1999) for detecting and quantifying bands. From the distribution of the RNA component among the protein-RNA and RNA bands in individual lanes on the gel, one can determine the equilibrium dissociation constant Kd of the complex in a straightforward manner. An approximate value for the equilibrium constant can be obtained by finding the concentration of protein at which roughly half the nucleic acid component is bound and half remains free. A more precise way to determine Kd is to plot individual fractions of the nucleic acid bound in each reaction versus the concentration of protein and then perform a nonlinear regression (Heffler, Walters, & Kugel, 2012) using free or commercially available software.

Human Argonaute 2 (Ago2) is an 859–amino acid protein that lies at the heart of the RNA- induced silencing complex (RISC; Sheu-Gruttadauria & MacRae, 2017). The protein binds small interfering RNA (siRNA) duplexes consisting of 21mer guide (antisense) and passenger (sense) strands with 3-terminal dinucleotide overhangs. Ago2 contains multiple domains, including the MID (binds 5-end of guide siRNA), PIWI (harbors the endonuclease active site), PAZ (binds 3-end of guide siRNA), and two linkage domains. The passenger strand is eventually cleaved or discarded and the target RNA loaded into Ago2 opposite the guide strand, resulting in cleavage of the former by the PIWI domain via a dual-metal ion mechanism (Watts & Corey, 2012; Wilson & Doudna, 2013). RISC contains other Ago proteins besides Ago2 (i.e., Ago1, Ago3, and Ago4), and while all are associated with micro RNAs (miRNAs), only Ago2 exhibits endonuclease activity (Meister et al., 2004). Chemically modified siRNAs are widely explored as therapeutics for treatment of a range of diseases (Crooke, Witztum, Bennett, & Baker, 2018; Shen & Corey 2018). ONPATTRO (Patisiran) is a modified siRNA formulated in lipids and manufactured by Alnylam Pharmaceuticals Inc. (Cambridge, MA) that was approved by the US FDA in August of 2018 for the treatment of polyneuropathy of hereditary transthyretin-mediated amyloidosis in adults (Sheridan, 2017). The PAZ domain of Ago2 binds the last two nucleotides of guide siRNA and assists in separating guide and passenger siRNA (Elkayam et al., 2012; Ma, Ye, & Patel, 2004; Schirle & MacRae, 2012; Fig. 1). The identities of the 3-overhanging nucleotides in the siRNA guide strand and their chemical modification affect the interaction with Ago2 PAZ and silencing activity (Alagia et al., 2018; Kandeel et al., 2014). We relied on the EMSA protocol presented here to determine equilibrium dissociation constants Kd of the interactions between native and chemically modified RNAs and the human Ago2 PAZ domain.

The following procedures outline the mixing of RNA and protein solutions of defined concentrations in various ratios to establish the binding reactions, the preparation of polyacrylamide gels to run EMSAs for analyzing protein-RNA binding, staining and imaging of gels to measure the intensities of individual bands and the distribution of RNA between the free and protein-bound forms, and the determination of Kd values using standard software to compute nonlinear regressions. However, neither the synthesis of RNA oligonucleotides nor the expression and purification of the Ago2 PAZ domain are described in detail. Briefly, RNAs were provided by Alnylam Pharmaceuticals Inc. (Cambridge, MA; native oligoribonucleotides) or AM Biotechnologies LLC (Houston, TX; 2-O-Me/3-phosphorodithioate [PS2; both non-bridging phosphate oxygen atoms Seo et al. replaced with sulfur] oligoribonucleotide, hereafter referred to as MS2-modified RNA). 2of12

Current Protocols in Nucleic Acid Chemistry Figure 1 Overlay of crystal structures of human Ago1- and Ago2-PAZ in complex with RNA oligonucleotides (A) and alignment of the human Ago1 (top line) and Ago2 (bottom line) sequences (B). Only a portion of the 859 amino acids is included; the consensus sequence is shown in the middle line with PAZ domain residues highlighted: 225 to 369 (Ago1) and 227 to 371 (Ago2). The PAZ domain binds the 3-end of guide siRNA. The models shown are from crystal structures of full-length Ago2 bound either to duplex RNA (Schirle & MacRae, 2012) or an miRNA single strand (Elkayam et al., 2012 ). In both cases, only PAZ domain residues 226 to 351 were included in the illustration. In both crystal structures, only portions of the 3-half of the RNA guide strand were visible in the electron density. The third structure depicted is of a complex between a separately expressed human Ago1 PAZ domain (residues 224 to 349 were resolved in the electron density) and an RNA nonamer (Ma et al., 2004). Coordinates were retrieved from the Protein Data Bank (PDB; https://www.rcsb.org/; Berman et al., 2000), and the structural ﬁgure in (A) was generated with the program UCSF Chimera (Pettersen et al., 2004).

Native RNAs were synthesized by standard or adapted solid phase phosphoramidite synthesis, purified (Andrus & Kuimelis, 2001b) by high-performance liquid chromatography (HPLC; Sinha & Jung, 2015), and desalted (Andrus & Kuimelis, 2001c). Identities and purities of all oligonucleotides were confirmed by electrospray ionization mass spectrom- etry (ESI-MS) and ion-exchange HPLC (IEX-HPLC), respectively. The MS2-modified RNA was synthesized and purified as reported in Yang (2017). An expression system for human Ago2 PAZ (amino acids 227 to 371; Fig. 1B) was generated by gene synthesis (GenScript Inc.) and subcloning into the pHD116 plasmid. The plasmid was transformed into E. coli BL21 (DE3) Gold cells using standard procedures. After expression the Seo et al. 3of12

Current Protocols in Nucleic Acid Chemistry protein was first purified by nickel affinity chromatography and, following cleavage of the His6 tag by PreScission protease (GE Healthcare), by ion exchange and gel filtra- tion chromatography. The identity of the Ago2 PAZ domain was established by tryptic digestion in combination with MS analysis. Materials 1% (w/v) agarose (see recipe) 5% (w/v) polyacrylamide gel solution (see recipe) 5× Tris/borate/EDTA (TBE) buffer (see recipe) 500 nM RNA(s) of interest (see Fig. 2 for sequences) Nuclease-free water (e.g., Promega, cat. no. P119E-C) Human Ago2 PAZ domain protein (see recipe) 1× binding buffer (see recipe) Bromophenol blue dye 25 mg/mL Ficoll (e.g., Sigma-Aldrich, cat. no. 26873-85-8) 10,000× Sybr Gold (e.g., Invitrogen, cat. no. S11494) Gel electrophoresis apparatus (e.g., Bio-Rad Mini-PROTEAN Tetra Cell System) containing: 1.5-mm-thick mini gel glass plates Gel combs Electrophoresis unit Power supply 1.5-mL microcentrifuge tubes Black cassettes Gel imaging system (e.g., Bio-Rad ChemiDoc MP Imaging System) Computer running image analysis software (e.g., ImageJ) Prepare and pre-run gel 1. Make a 5% native TBE polyacrylamide gel by first assembling two 1.5-mm-thick glass gel plates (i.e., with clips), and set into the gel apparatus. 2. Seal the bottom of the gel plates with 1% agarose using a 1.0-mL micropipette. Be sure to pipette agarose on both sides of the gel plates to seal them as well. 3. Prepare 10 mL of 5% polyacrylamide gel solution (0.5× TBE). 4. Using a 1.0-mL micropipette, inject the polyacrylamide solution into the gel cast. Insert the comb into the assembled gel sandwich. Wait 1 hr to let the gel polymerize.

Figure 2 Gel images of binding reactions with various ratios between Ago2 PAZ and (A) native RNA and (B) MS2-modiﬁed RNA. RNA sequences are shown below the gels, and protein Seo et al. concentrations are shown above individual lanes. The RNA concentration was 250 nM. 4of12

Current Protocols in Nucleic Acid Chemistry Table 1 Sample Volumes and Concentrations of PAZ and RNA Needed for ESMA

Protein PAZ protein 1× binding RNA(s) of concentration (nM) added (µL) buffer added (µL) interest added (µL) 500 1.0 (10 µM stock) 9.0 10.0 (500 nM stock) 600 1.2 8.8 10.0 700 1.4 8.6 10.0 900 1.6 8.2 10.0 1250 2.5 7.5 10.0 2000 4.0 6.0 10.0 2500 5.0 5.0 10.0 3000 6.0 4.0 10.0 6000 3.0 (40 µM stock) 7.0 10.0

5. Prepare electrophoresis running buffer by diluting 100 mL of 5× TBE buffer with 900 mL sterile water to create 1 liter of 0.5× TBE. 6. Pre-run the polyacrylamide gel by submerging the cast gel in the running buffer from step 5, and run at 45 V to 60 V for 1 hr or until the current is stable. Prepare sample and run and stain the gel 7. Prepare 100 μL of 500 nM RNA stock per gel in nuclease-free water, and dilute PAZ protein with 1× binding buffer to create two protein stock solutions, one of 10 μM concentration and the other of 40 μM concentration. 8. Allow the stock solutions prepared in step 7 to warm to room temperature, and then prepare nine human Ago2 PAZ protein and RNA binding mixtures in separate 1.5-mL microcentrifuge tubes according to Table 1. Create a control sample by mixing 10 μLRNAand10μL bromophenol blue dye. Incubate at room temperature for 30 min. 9. After the incubation is complete, add 2 μL Ficoll solution to each tube, including the control. 10. Load 20 μL of each sample into a well with the gel. Load the control sample into the ﬁrst lane. 11. Run the gel at 40 to 70 V, 6 to 15 mA, for 1 hr or until the samples are roughly two-thirds down the gel (Fig. 2). 12. Add 5 μL of 10,000× Sybr Gold to 50 mL of 0.5× TBE buffer to create 50 mL of 1× Sybr Gold solution. 13. Stain each gel by submerging it in 50 mL of 1× Sybr Gold in a black cassette for 30 min. The Sybr Gold stain does not bind to the Ago2 PAZ domain. 14. Rinse gels with deionized water (optional). 15. Image gel using a gel imaging system (e.g., ChemiDoc MP Imaging System with a Blot/UV/Stain-Free Sample Tray; http://www.bio-rad.com/en-us/product/chemi doc-mp-imaging-system?ID=NINJ8ZE8Z; Fig. 2). 16. Calculate the binding afﬁnity between the RNA and the protein by analyzing the gel images with ImageJ. See the Anticipated Results section for more details. Seo et al. 5of12

Current Protocols in Nucleic Acid Chemistry REAGENTS AND SOLUTIONS Agarose, 1% 0.5 g agarose 50 mL 0.5× TBE buffer (see recipe) Store at 4°C for up to 2 weeks Ammonium persulfate (APS) solution, 10% 0.1 g APS (e.g., Bio-Rad, cat. no. 161-0700) 1 mL sterile water Prepare freshly before use Binding buffer, 1× 1mL10× binding buffer (see recipe) 2 mL 50% glycerol (e.g., RPI Corp., cat. no. 56-81-5) 7 mL sterile water Store at 4°C for up to 2 weeks Binding buffer, 10× 0.1 mM HEPES 1.5 M NaCl (e.g., Acros Organics, cat. no. 7647-14-5) 30 mM EDTA (e.g., Sigma-Aldrich, cat.no. 60-00-4) 10 mM DTT (e.g., Sigma-Aldrich, cat. no. 3483-12-3) Store at 4°C for up to 2 weeks Human Ago2 PAZ domain protein 180 μM PAZ protein 5 mM HEPES 100 mM KCl 10 mM DTT (e.g., Sigma-Aldrich, cat. no. 3483-12-3) Store at −80°C for up to 1 year Polyacrylamide gel solution, 5% 7.27 mL sterile water 1.66 mL 30% acrylamide/bis solution 37.5:1 (e.g., Bio-Rad, cat. no. 161-0158) 1.00 mL 5× TBE (see recipe) 70 mL 10% APS (see recipe) 3.5 mL tetramethylethylenediamine (TEMED; e.g., Bio-Rad, cat. no. 161-0800) Prepare freshly before use TBE buffer, 5× 1 liter water 54 g Tris base (e.g., RPI Corp., cat. no. 77-86-1) 27.5 g boric acid (e.g., RPI Corp., cat. no. 10043-35-3) 20 mL 0.5 M EDTA (e.g., Sigma-Aldrich, cat. no. 60-00-4), pH 8.0 Store at room temperature for up to 2 months To prepare 0.5× TBE buffer, dilute 100 mL of 5× TBE buffer with 900 mL sterile water.

COMMENTARY Background Information Chen, 2011; Fried, 1989; Heffler et al., 2012; There are multiple techniques for determin- Hellman & Fried, 2007). These include the ing the equilibrium dissociation constant Kd filter-binding assay (Woodbury & von Hip- of a protein–nucleic acid binding interaction pel, 1983), fluorescence intensity (Teplova Seo et al. besides the EMSA (Alves & Cunha, 2012; et al., 2000), chemical shift differences using 6of12

Current Protocols in Nucleic Acid Chemistry 1H–15N heteronuclear single quantum coher- ues for the stability of the complex. Thus, the ence (HSQC) nuclear magnetic resonance Kd values for the interactions between Ago2 (NMR) titration experiments (Frank, Hau- PAZ and mono- (e.g., UMP) and dinucleotides ver, Sonenberg, & Bushan, 2012), surface (e.g., UpUMP) varied between 10 and 60 μM plasmon resonance (SPR; Ma et al., 2004; based on ITC (Kandeel et al., 2014). In con- McDonnell, 2001), acoustic measurements trast, the Kd of the interaction between Ago1 (Cooper & Whalen, 2005; Godber et al., PAZ and an 11mer/13mer RNA duplex with 2005), biolayer interferometry (BLI; Lou, a3-terminal dinucleotide overhang (Fig. 2A) Egli, & Yang, 2016), microscale thermophore- was reported to be 2.2 nM as determined by sis (MST; Mueller et al., 2017), and isothermal SPR (Ma et al., 2004; Ago1 and Ago2 PAZ titration calorimetry (ITC; Rozners, Pilch, & have highly similar sequences; Fig. 1B). In or- Egli, 2015), among others. der to verify the EMSA-based Kd values, we The choice to use one method versus therefore decided to also conduct independent another may depend on the amount of mate- ITC experiments. The outcomes of the two rial available, the particular environment in approaches are discussed in the Anticipated which the experiment is to be conducted (e.g., Results section. solution, chip, microfluidics, cell), whether labeling can be accomplished relatively easily and/or cheaply, or whether equipment that is Critical Parameters and needed with some of the above techniques is Troubleshooting accessible to the investigator. Thus, EMSAs, We have tested two types of polyacrylamide spectroscopic approaches, and SPR require gels and varied their percentages. less material than filter-binding assays and (1) TBE polyacrylamide gel:Westarted ITC. An important consideration concerns the from 15% and then continuously lowered the Kd limit, that is the upper end of the stability percentage to 12%, 10%, 8%, 5%, and finally range for the complex between protein and to 3.5%, as gels of higher percentage poly- nucleic acid of interest at which a particular acrylamide prevented the complex from get- technique still affords precise data. In this ting into and migrating through the gel. How- regard, EMSA, SPR, and BLI allow reliable ever, a 3.5% gel turned out to be too frail to measurements of interactions with Kd values handle, and it also showed no significant im- of as low as 10−12 M (Jing & Bowser, 2011). provement compared to the 5% gel in terms of This contrasts with the Kd limit for ITC that is the migration of the complex. Thus, a 5% gel around 10−8 to 10−9 M. However, ITC, despite was judged to be optimal in terms of structural this limitation and the need for relatively large integrity and migration of the complex, even amounts of material, offers the benefits of a at higher concentrations of PAZ protein. solution environment and label-free binding (2) Tris-glycine polyacrylamide gel:We partners; it is also the method of choice to assayed binding reactions on 4% to 20%, 12%, establish precise thermodynamic parameters 10%, and 7.5% gels. Although the complex of a binding interaction (ΔH, ΔS, and ΔG; band migrated through the gel, increasing the Rozners et al., 2015). Most of the alternative protein:RNA ratio did not result in reduced and approaches that yield the Kd of an interaction increased intensity of the RNA and complex require labeling of either protein or nucleic band, respectively, and such gels apparently acid, such as 15N (protein; NMR), 32P(DNA do not constitute a suitable environment for or RNA; filter-binding assay, EMSA), biotiny- the PAZ-RNA complex. lation (DNA or RNA; SPR, BLI; Lou et al., We also examined the effects of varying 2016), or fluorescent probes (protein or nu- the pH (e.g., 1% TBE, pH 8.0, and 0.5% TBE, cleic acid; MST, EMSA; Jiang & Egli, 2011). pH 7.6) and the addition of detergents (0.04% We opted to use EMSA with label-free olig- sodium cholate, 1/10 CMC; 0.008% Triton X- oribonucleotides to determine the Kd of the in- 100, 1/2 CMC). However, detergents proved teraction between human Ago2 PAZ and RNA. to be detrimental to the complex. Thus, a 0.5× Advantages of this approach are speed, low TBE buffer for both gel and running buffer cost, and the small amount of material needed. yielded much better results. We did not use + + A familiar limitation lies in the environment Mg2 or Ca2 in the buffer, and neither was (i.e., a gel matrix and not solution) that can po- used for annealing the RNAs. We do not an- tentially affect the binding reaction. The com- ticipate that the presence of these ions would plex between PAZ domain and RNA is known have a significant effect. Moreover, neither ion is present in or near the binding pocket of the to be of a 1:1 stoichiometry (Fig. 1A), but Seo et al. published data reveal somewhat divergent val- PAZ domain. 7of12

Current Protocols in Nucleic Acid Chemistry Figure 3 Nonlinear regressions for the PAZ complex with (A) native RNA and (B) modiﬁed RNA based on the gels depicted in Figure 2.

We observed that gel bands representing est RID being subtracted from itself resulting the PAZ-RNA complex tended to smear and in zero. These values are the relative RIDs. that migration was limited at the highest ra- Each relative RID was divided by the largest tio between protein and RNA. This issue may relative RID and then multiplied by 100, with become exacerbated by the use of higher con- the largest relative RID being divided by it- centrations of protein and RNA owing to the self and then multiplied by 100, resulting in lack of labels on the latter. However, the RNA 100. These are the percentage RIDs. Finally, bands were typically quite sharp, allowing rea- each percentage RID was subtracted from 100, sonably precise density measurements even in resulting in the percentage of binding (i.e., cases where migration of the complex was lim- complex formation, for each protein concen- ited or bands representing the complex were tration). spread out. These results then served as input for For PAGE experiments one can of course GraphPad Prism (version 5.00 for Mac; Graph- use either precast or handcast gels. We have Pad Software, La Jolla, CA; https://www. relied on commercial precast gels in some graphpad.com/). Select the option that plots a cases but have also poured our own gels. single “Y” value for each data point, and press There are various systems on the market that create. Enter the protein concentrations in the facilitate hand pouring gels, (e.g., Sure Cast x-column, and enter the percent binding in the Gel Handcast System offered by Thermo y-column. Label both columns. Next, click on Fisher; https://www.thermofisher.com/us/en/ the results tab, and select nonlinear regression home/life-science/protein-biology/protein-gel- (curve fit) under “XY analyses.” Then select electrophoresis/protein-gels/surecast-gel-hand the equation that is labeled “one site–specific cast-system.html). binding,” which computes the Kd value (Fig. 3). The Kd values obtained for the PAZ com- Anticipated Results plexes with native and modified RNA are To analyze the gel image obtained, the NIH shown in Table 2. program ImageJ was used (Schneider, Ras- Our EMSA is relatively simple, cheap, band, & Eliceiri, 2012). The program can be and quick. However, we wanted to deter- downloaded from the NIH website for free mine if it also provided reasonably accurate (https://imagej.nih.gov/ij). The image shown data, keeping in mind the usual limitations in Figure 2 was first inverted, and then the of a gel-based assay. We therefore decided to raw integrated density (RID) of each band was compare the EMSA-based data to K values measured with ImageJ. ImageJ may not have d obtained from ITC experiments. These Kd val- the RID as a default setting. If that is the case, ues were 0.6 μM (PAZ complex with native go to the “Analyze” tab, click “Set Measure- RNA; Fig. 4A) and 1.9 μM (PAZ complex ments,” check the “Integrated Density” box, with modified RNA; Fig. 4B) and therefore and then click “OK.” The results were then quite similar to the EMSA data. Typically, one transferred into an Excel spreadsheet to per- would carry out the EMSA in triplicate and form the following calculations. Each RID was Seo et al. then report average values. subtracted from the lowest RID, with the low- 8of12

Current Protocols in Nucleic Acid Chemistry Table 2 Kd Values Obtained for PAZ Complexes with Native and Modiﬁed RNA

RNA Kd [µM], nonlinear ﬁt 5–AGA GUC ACG CUU U–3 1.7 3–UCU CAG UGC GA–5 5 –AGA GUC ACG Cms2Ums2UU–3 3.9 3–UCU CAG UGC G A –5

Figure 4 Results of isothermal titration calorimetry experiments for the PAZ complex with (A) native RNA and (B) modiﬁed RNA. The experiments were conducted under the following conditions: 56 μM Ago2 PAZ and 600 μM RNA in a buffer composed of 5 mM HEPES, pH 7.5, 150 mM KCl, 10% glycerol, and 10 mM DTT. RNA was added to the protein solution using 28 to 33 1.2-μL injections at 25°C. Note the 1:1 stoichiometry of the complexes, consistent with the structural models depicted in Figure 1A.

An earlier publication had reported a & Sullenger, 2008; PDB ID 3DD2) that ex- 2.2 nM Kd for the Ago1 PAZ complex with hibits a Kd of 1 nM (Abeydeera et al., 2016), the same native RNA construct that was em- protein 719 A˚ 2, and RNA 790 A˚ 2 is much ployed here and using SPR (Ma et al., 2004). larger than that between Ago2 PAZ and the 3- However, we believe that this may be an over- end of an RNA (complex with PDB ID 4F3T; estimation of the tightness of binding between Fig. 1A): protein 344 A˚ 2 and RNA 465 A˚ 2. PAZ domain and RNA. For one, Ago1 and However, it is of note that complexes can ex- Ago2 PAZ have similar sequences (Fig. 1B), hibit dramatically different stabilities as a con- and it is unlikely that the former should ex- sequence of a key interaction that leaves the ceed the latter in terms of its afﬁnity for buried surface essentially unchanged. Thus, RNA by three orders of magnitude. In ad- replacement of a single phosphate (PO2) by dition, the buried surface (binding interface, a phosphorodithioate (PS2) moiety in RNA calculated with the program PRince; Barik, aptamers was reported to trigger a dramatic Mishra, & Bahadur, 2012; http://www.facweb. change in the dissociation constant of the iitkgp.ac.in/rbahadur/prince/home.html)in complexes with their respective targets from a complex between thrombin and an anti- about 1 nM to about 1 pM (anti-thrombin and thrombin RNA aptamer (Long, Long, White, anti-VEGF aptamers; Abeydeera et al., 2016). Seo et al. 9of12

Current Protocols in Nucleic Acid Chemistry Finally, the Kd values for complexes between https://www.intechopen.com/books/gel-electrop PIWI protein PAZ domains and various RNAs horesis-advanced-techniques/electrophoretic- based on ITC were recently reported to be be- mobility-shift-assay-analyzing-protein-nucleic- acid-interactions. tween 2 and 34 μM (Tian, Simanshu, Ma, & Patel, 2011). Andrus, A., & Kuimelis, R. G. (2001a). Poly- acrylamide gel electrophoresis (PAGE) of synthetic nucleic acids. Current Protocols in Nu- cleic Acid Chemistry, 1, 10.4.1–10.4.10. doi: Time Considerations 10.1002/0471142700.nc1004s01. The entire experiment, from pouring the Andrus, A., & Kuimelis, R. G. (2001b), Overview gels to imaging and calculating the Kd value of purification and analysis of synthetic takes around 6 hr. Pouring, preparing, waiting nucleic acids. Current Protocols in Nu- for the gel to polymerize, and pre-running the cleic Acid Chemistry, 1, 10.3.1–10.3.6. doi: gel can be accomplished in around 2 hr. Do- 10.1002/0471142700.nc1003s01. ing the calculations to prepare the individual Andrus, A., & Kuimelis, R. G. (2001c). Car- samples, preparing the samples, loading the tridge methods for oligonucleotide purification. samples, and running the gel takes 2 to 3 hr. Current Protocols in Nucleic Acid Chemistry, 1, 10.7.1–10.7.5. doi: 10.1002/0471142700. Finally, gel imaging, measuring band densi- nc1007s01. ties with ImageJ, and obtaining the K value d Barik, A., Mishra, A., & Bahadur, R. P. (2012). using Prism takes another hour. With one gel PRince: A web server for structural and physic- apparatus, two gels can be run simultaneously, ochemical analysis of protein-RNA interface. and thus Kd values for two complexes can be Nucleic Acids Research, 40, W440–W444. doi: obtained on the same day. 10.1093/nar/gks535. Berman, H. 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